荆芥挥发油抗内毒素中毒小鼠NLRP3炎症小体通路的机制研究
|
摘要
目的以NLRP3炎症小体通路为切入点,探究荆芥挥发油抗炎效应的分子机制。方法荆芥挥发油低、高剂量(0.226、0.452 g·kg~(-1))连续灌胃给药5 d,末次给药后30 min,小鼠腹腔注射LPS (0.015 g·kg~(-1),10 mL·kg~(-1))构建内毒素中毒小鼠模型,造模后12 h取材,测定相关指标。Griess法测定肺组织NO含量;qPCR法检测肺组织中NLRP3、ASC、caspase-1、IL-1β、iNOS、p65 mRNA的表达;免疫组化法检测肺组织中P2X7R、Cathepsin B的表达;Western blot法测定肺组织中NLRP3、caspase-1(p20)、pro-IL-1β、COP1蛋白表达。结果 0.226 g·kg~(-1)荆芥挥发油明显降低模型小鼠肺组织Cathepsin B、NLRP3蛋白表达;0.452 g·kg~(-1)荆芥挥发油明显降低模型小鼠肺组织中NO水平,明显下调小鼠肺组织中NLRP3、iNOS、p65、IL-1βmRNA的表达及P2X7R蛋白表达;荆芥挥发油(0.226、0.452 g·kg~(-1))均能明显下调caspase-1(p20)蛋白水平,升高COP1蛋白水平。结论荆芥挥发油对内毒素中毒小鼠的保护作用与其抗炎效应密切相关,抗炎机制与抑制NLRP3炎症小体的激活有关。
Aim To explore the mechanism of the anti-inflammatory effect of essential oils of Schizonepeta tenuifolia Briq.(EOST) by investigating the regulatory effect on NLRP3 inflammasome pathway. Methods EOST(0.226, 0.452 g·kg~(-1)) were continuously administered for five days. Thirty minutes after the last administration, the mice were intraperitoneally injected with LPS(0.015 g·kg~(-1), 10 mL·kg~(-1)) to construct endotoxin poisoning, and relevant indicators for sample determination were taken 12 h after modeling. Determination of NO level in lung tissues by Griess method was performed. The expressions of NLRP3, ASC, caspase-1, IL-1β, iNOS and p65 mRNA in lung tissues were detected by qPCR. The expressions of P2 X7 R and Cathepsin B protein in lung tissues were assessed using immunohistochemistry. The NLRP3, caspase-1(p20), Pro-caspase-1, COP-1 protein expressions were detected by Western blot. Results 0. 226 g·kg~(-1) EOST could down-regulate the expressions of Cathepsin B and NLRP3 proteins in lung tissues of mice; 0. 452 g · kg~(-1) EOST significantly reduced NO level,the expressions of NLRP3,i NOS,p65,IL-1β mRNA and P2 X7 R protein in lung tissues of mice; EOST( 0. 226,0. 452 g·kg~(-1)) markedly reduced the caspase-1( p20) protein level and significantly increased COP1 protein level. Conclusions The protective effect of EOST on endotoxin poisoning mice is closely related to its anti-inflammatory effects,which involves the inhibition of activation of NLRP3 inflammasome.
Aim To explore the mechanism of the anti-inflammatory effect of essential oils of Schizonepeta tenuifolia Briq.(EOST) by investigating the regulatory effect on NLRP3 inflammasome pathway. Methods EOST(0.226, 0.452 g·kg~(-1)) were continuously administered for five days. Thirty minutes after the last administration, the mice were intraperitoneally injected with LPS(0.015 g·kg~(-1), 10 mL·kg~(-1)) to construct endotoxin poisoning, and relevant indicators for sample determination were taken 12 h after modeling. Determination of NO level in lung tissues by Griess method was performed. The expressions of NLRP3, ASC, caspase-1, IL-1β, iNOS and p65 mRNA in lung tissues were detected by qPCR. The expressions of P2 X7 R and Cathepsin B protein in lung tissues were assessed using immunohistochemistry. The NLRP3, caspase-1(p20), Pro-caspase-1, COP-1 protein expressions were detected by Western blot. Results 0. 226 g·kg~(-1) EOST could down-regulate the expressions of Cathepsin B and NLRP3 proteins in lung tissues of mice; 0. 452 g · kg~(-1) EOST significantly reduced NO level,the expressions of NLRP3,i NOS,p65,IL-1β mRNA and P2 X7 R protein in lung tissues of mice; EOST( 0. 226,0. 452 g·kg~(-1)) markedly reduced the caspase-1( p20) protein level and significantly increased COP1 protein level. Conclusions The protective effect of EOST on endotoxin poisoning mice is closely related to its anti-inflammatory effects,which involves the inhibition of activation of NLRP3 inflammasome.
引文
[1] 曾南, 沈映君, 刘旭光, 等. 荆芥挥发油抗炎作用研究[J]. 中药药理与临床,1998,14(6):24-8.[1] Zeng N, Shen Y J, Liu X G, et all. Studies on the anti-inflammatory effect of Jingjie volatile oil[J]. Pharmacol Clin Chin Mater Med, 1998, 14(6):24-8.
[2] 温桃群, 桑文涛, 徐锋, 等. 荆芥挥发油对内毒素中毒模型小鼠的保护作用[J].中国中药杂志,2016,41(24):4642-7.[2] Wen T Q, Sang W T, Xu F, et all. Protective effects of Schizonepeta volatile oil on endotoxin poisoning induced by lipopolysaccharide in mice[J]. Chin J Chin Mater Med, 2016, 41(24):4642-7.
[3] 侯丽. 一种特殊的生物信使分子一氧化氮[J]. 齐齐哈尔医学院学报,1999,20(4):402-3.[3] Hou L. A kind of special organismic massager molecular—nitrogen monoxide[J]. J Qiqihar Med Coll, 1999, 20(4):402-3.
[4] Gaston B, Drazen J M, Loscalzo J, et al. The biology of nitrogen oxides in the airways[J]. Am J Respir Crit Care Med, 1994, 149(2 Pt 1):538.
[5] 范精华, 刘康, 刘保林. NO在炎症及免疫应答中的调节作用[J]. 中外医疗, 2009,28(25):163,166.[5] Fan J H, Liu K, Liu B L. Regulation of NO in inflammation and immune response[J]. Chin Foreign Med Treatment, 2009, 28(25):163,166.
[6] 王凤,温桃群,徐锋,等. 薄荷酮对内毒素致炎症模型小鼠的保护作用研究[J].中国药理学通报,2017,33(2):227-34.[6] Wang F, Wen T Q, Xu F, et al. The protective effects of menthone on endotoxin-induced inflammation in mice[J]. Chin Pharmacol Bull, 2017, 33(2):227-34.
[7] Si M M, Kanneganti T D. Gasdermin D: the long-awaited executioner of pyroptosis[J]. Cell Res, 2015, 25(11):1183-4.
[8] 景艳芸,李陈广,颜亮,等. 灯盏花乙素对J774A.1巨噬细胞中ATP诱导的炎症小体活化和细胞焦亡的影响[J]. 中国药理学通报,2018,34(2):174-80.[8] Jing Y Y, Li C G, Yan L, et al. Influences of scutellarin on ATP-induced inflammasome activation and pyroptosis in J774A.1 macrophages[J]. Chin Pharmacol Bull, 2018, 34(2):174-80.
[9] Muňoz-Planillo R, Kuffa P, Martínez-Colón G, et al. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter[J]. Immunity, 2013, 38(6):1142-53.
[10] Lamkanfi M, Dixit V M. Inflammasomes and their roles in health and disease[J]. Annu Rev Cell Dev Biol, 2012, 28(28):137-61.
[11] Mezzasoma L, Antognelli C, Talesa V N. Atrial natriuretic peptide down-regulates LPS/ATP-mediated IL-1β release by inhibiting NF-κB, NLRP3 inflammasome and caspase-1 activation in THP-1 cells[J]. Immunol Res, 2016, 64(1):303-12.
[12] Stehlik C, Dorfleutner A. COPs & POPs: modulators of inflammasome activity[J]. J Immunol, 2007, 179(12):7993-8.
[2] 温桃群, 桑文涛, 徐锋, 等. 荆芥挥发油对内毒素中毒模型小鼠的保护作用[J].中国中药杂志,2016,41(24):4642-7.[2] Wen T Q, Sang W T, Xu F, et all. Protective effects of Schizonepeta volatile oil on endotoxin poisoning induced by lipopolysaccharide in mice[J]. Chin J Chin Mater Med, 2016, 41(24):4642-7.
[3] 侯丽. 一种特殊的生物信使分子一氧化氮[J]. 齐齐哈尔医学院学报,1999,20(4):402-3.[3] Hou L. A kind of special organismic massager molecular—nitrogen monoxide[J]. J Qiqihar Med Coll, 1999, 20(4):402-3.
[4] Gaston B, Drazen J M, Loscalzo J, et al. The biology of nitrogen oxides in the airways[J]. Am J Respir Crit Care Med, 1994, 149(2 Pt 1):538.
[5] 范精华, 刘康, 刘保林. NO在炎症及免疫应答中的调节作用[J]. 中外医疗, 2009,28(25):163,166.[5] Fan J H, Liu K, Liu B L. Regulation of NO in inflammation and immune response[J]. Chin Foreign Med Treatment, 2009, 28(25):163,166.
[6] 王凤,温桃群,徐锋,等. 薄荷酮对内毒素致炎症模型小鼠的保护作用研究[J].中国药理学通报,2017,33(2):227-34.[6] Wang F, Wen T Q, Xu F, et al. The protective effects of menthone on endotoxin-induced inflammation in mice[J]. Chin Pharmacol Bull, 2017, 33(2):227-34.
[7] Si M M, Kanneganti T D. Gasdermin D: the long-awaited executioner of pyroptosis[J]. Cell Res, 2015, 25(11):1183-4.
[8] 景艳芸,李陈广,颜亮,等. 灯盏花乙素对J774A.1巨噬细胞中ATP诱导的炎症小体活化和细胞焦亡的影响[J]. 中国药理学通报,2018,34(2):174-80.[8] Jing Y Y, Li C G, Yan L, et al. Influences of scutellarin on ATP-induced inflammasome activation and pyroptosis in J774A.1 macrophages[J]. Chin Pharmacol Bull, 2018, 34(2):174-80.
[9] Muňoz-Planillo R, Kuffa P, Martínez-Colón G, et al. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter[J]. Immunity, 2013, 38(6):1142-53.
[10] Lamkanfi M, Dixit V M. Inflammasomes and their roles in health and disease[J]. Annu Rev Cell Dev Biol, 2012, 28(28):137-61.
[11] Mezzasoma L, Antognelli C, Talesa V N. Atrial natriuretic peptide down-regulates LPS/ATP-mediated IL-1β release by inhibiting NF-κB, NLRP3 inflammasome and caspase-1 activation in THP-1 cells[J]. Immunol Res, 2016, 64(1):303-12.
[12] Stehlik C, Dorfleutner A. COPs & POPs: modulators of inflammasome activity[J]. J Immunol, 2007, 179(12):7993-8.